Proof of concept for multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Abstract

Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies’ MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×–19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.

An Evaluation of Avian Influenza Virus Whole Genome Sequencing Approaches Using Nanopore Technology

“…When the reads in the region near the premature termination codon were examined, a frameshift caused by missing a nucleotide in a run of homopolymers was frequently found. For the MinION platform, 60-fold or higher read coverage has been found to result in a better than 99.95% sequencing accuracy [29] [30]. In our study, Method K resulted in the identical sequence in two samples (245467 and 246038) when compared to the sequence generated from the same samples on the MiSeq (Supplementary Figures 2a-h & 4a-h).…”

Mitochondrial Genetic Diversity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Associated with Cassava in Lao PDR

“…Although the main problem with nanopore sequencing is its still relatively high per-base error rate in raw reads compared to other sequencing techniques, these are offset by the high coverages obtained and good bioinformatics handling [ 56 ]. Previous reports indicate that a 100× coverage is adequate to establish an accurate genotype; furthermore, advances in nanopore sequencing technology, in combination with a dedicated data analysis pipeline and good coverage, have proven to be comparable in performance to conventional Sanger sequencing applied to PCR amplicons, as is the case in this work [ 57 , 58 ].…”

Evaluation ofMycobacterium Tuberculosisenrichment in metagenomic samples using ONT adaptive sequencing and amplicon sequencing for identification and variant calling

“…Taxonomic classification with the amplicon data can be challenging even with longer amplicons 24 , which is especially useful for lineage tracing 25 . In addition, amplification efficiency among different amplicon regions might vary significantly, depending on the sequence complexity length of the amplicon and GC content 26 . In our study, among the 19 targeted regions, Rv0678 showed the highest amplification efficiency (with 981,074 coverage), while rspA showed the lowest (with 166.522 coverage) ( Supplementary Table 1 ).…”